Showing papers on "Microsatellite published in 2015"

Analysis of Population Structure and Genetic Diversity in Rice Germplasm Using SSR Markers: An Initiative Towards Association Mapping of Agronomic Traits in Oryza Sativa

Abstract: Genetic diversity is the main source of variability in any crop improvement program. It serves as a reservoir for identifying superior alleles controlling key agronomic and quality traits through allele mining/association mapping. Association mapping based on LD (Linkage dis-equilibrium), non-random associations between causative loci and phenotype in natural population is highly useful in dissecting out genetic basis of complex traits. For any successful association mapping program, understanding the population structure and assessing the kinship relatedness is essential before making correlation between superior alleles and traits. The present study was aimed at evaluating the genetic variation and population structure in a collection of 192 rice germplasm lines including local landraces, improved varieties and exotic lines from diverse origin. A set of 192 diverse rice germplasm lines were genotyped using 61 genome wide SSR markers to assess the molecular genetic diversity and genetic relatedness. Genotyping of 192 rice lines using 61 SSRs produced a total of 205 alleles with the PIC value of 0.756. Population structure analysis using model based and distance based approaches revealed that the germplasm lines were grouped into two distinct subgroups. AMOVA analysis has explained that 14 % of variation was due to difference between with the remaining 86 % variation may be attributed by difference within groups. Based on these above analysis viz., population structure and genetic relatedness, a core collection of 150 rice germplasm lines were assembled as an association mapping panel for establishing marker trait associations.

High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA

Abstract: High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This sequence structure can be harnessed to improve bioinformatics algorithms, in particular for CNV and structural variant detection. Descriptive measures for cell-free DNA features developed here could also be used in biomarker analysis to monitor the changes that occur during different pathological conditions.

Performance of a next generation sequencing SNP assay on degraded DNA.

Abstract: Forensic DNA casework samples are often of insufficient quantity or quality to generate full profiles by conventional DNA typing methods. Polymerase chain reaction (PCR) amplification of short tandem repeat (STR) loci is inherently limited in samples containing degraded DNA, as the cumulative size of repeat regions, primer binding regions, and flanking sequence is necessarily larger than the PCR template. Additionally, traditional capillary electrophoresis (CE) assay design further inherently limits shortening amplicons because the markers must be separated by size. Non-traditional markers, such as single nucleotide polymorphisms (SNPs) and insertion deletion polymorphisms (InDels), may yield more information from challenging samples due to their smaller amplicon size. In this study, the performance of a next generation sequencing (NGS) SNP assay and CE-based STR, mini-STR, and InDel assays was evaluated with a series of fragmented, size-selected samples. Information obtained from the NGS SNP assay exhibited higher overall inverse random match probability (1/RMP) values compared to the CE-based typing assays, with particular benefit for fragment sizes ≤ 150 base pairs (bp). The InDel, mini-STR, and NGS SNP assays all had similar percentages of loci with reportable alleles at this level of degradation; however, the relatively fewer number of loci in the InDel and mini-STR assays results in the NGS SNP assay having at least nine orders of magnitude higher 1/RMP values. In addition, the NGS SNP assay and three CE-based assays (two STR and one InDel assay) were tested using a dilution series consisting of 0.5 ng, 0.1 ng, and 0.05 ng non-degraded DNA. All tested assays showed similar percentages of loci with reportable alleles at these levels of input DNA; however, due to the larger number of loci, the NGS SNP assay and the larger of the two tested CE-based STR assays both resulted in considerably higher 1/RMP values than the other assays. These results indicate the potential advantage of NGS SNP assays for forensic analysis of degraded DNA samples.

A comparison of single nucleotide polymorphism and microsatellite markers for analysis of parentage and kinship in a cooperatively breeding bird

Abstract: The development of genetic markers has revolutionized molecular studies within and among populations. Although poly-allelic microsatellites are the most commonly used genetic marker for within-population studies of free-living animals, biallelic single nucleotide polymorphisms, or SNPs, have also emerged as a viable option for use in nonmodel systems. We describe a robust method of SNP discovery from the transcriptome of a nonmodel organism that resulted in more than 99% of the markers working successfully during genotyping. We then compare the use of 102 novel SNPs with 15 previously developed microsatellites for studies of parentage and kinship in cooperatively breeding superb starlings (Lamprotornis superbus) that live in highly kin-structured groups. For 95% of the offspring surveyed, SNPs and microsatellites identified the same genetic father, but only when behavioural information about the likely parents at a nest was included to aid in assignment. Moreover, when such behavioural information was available, the number of SNPs necessary for successful parentage assignment was reduced by half. However, in a few cases where candidate fathers were highly related, SNPs did a better job at assigning fathers than microsatellites. Despite high variation between individual pairwise relatedness values, microsatellites and SNPs performed equally well in kinship analyses. This study is the first to compare SNPs and microsatellites for analyses of parentage and relatedness in a species that lives in groups with a complex social and kin structure. It should also prove informative for those interested in developing SNP loci from transcriptome data when published genomes are unavailable.

Identification and analysis of novel salt responsive candidate gene based SSRs (cgSSRs) from rice ( Oryza sativa L.)

Abstract: Majority of the Asian people depend on rice for nutritional energy. Rice cultivation and yield are severely affected by soil salinity stress worldwide. Marker assisted breeding is a rapid and efficient way to develop improved variety for salinity stress tolerance. Genomic microsatellite markers are an elite group of markers, but there is possible uncertainty of linkage with the important genes. In contrast, there are better possibilities of linkage detection with important genes if SSRs are developed from candidate genes. To the best of our knowledge, there is no such report on SSR markers development from candidate gene sequences in rice. So the present study was aimed to identify and analyse SSRs from salt responsive candidate genes of rice. In the present study, based on the comprehensive literature survey, we selected 220 different salt responsive genes of rice. Out of them, 106 genes were found to contain 180 microsatellite loci with, tri-nucleotide motifs (56%) being most abundant, followed by di-(41%) and tetra nucleotide (2.8%) motifs. Maximum loci were found in the coding sequences (37.2%), followed by in 5′UTR (26%), intron (21.6%) and 3′UTR (15%). For validation, 19 primer sets were evaluated to detect polymorphism in diversity analysis among the two panels consisting of 17 salt tolerant and 17 susceptible rice genotypes. Except one, all primer sets exhibited polymorphic nature with an average of 21.8 alleles/primer and with a mean PIC value of 0.28. Calculated genetic similarity among genotypes was ranged from 19%-89%. The generated dendrogram showed 3 clusters of which one contained entire 17 susceptible genotypes and another two clusters contained all tolerant genotypes. The present study represents the potential of salt responsive candidate gene based SSR (cgSSR) markers to be utilized as novel and remarkable candidate for diversity analysis among rice genotypes differing in salinity response.

Genetic diversity and relationship of Indian cattle inferred from microsatellite and mitochondrial DNA markers

Abstract: Indian agriculture is an economic symbiosis of crop and livestock production with cattle as the foundation. Sadly, the population of indigenous cattle (Bos indicus) is declining (8.94 % in last decade) and needs immediate scientific management. Genetic characterization is the first step in the development of proper management strategies for preserving genetic diversity and preventing undesirable loss of alleles. Thus, in this study we investigated genetic diversity and relationship among eleven Indian cattle breeds using 21 microsatellite markers and mitochondrial D loop sequence. The analysis of autosomal DNA was performed on 508 cattle which exhibited sufficient genetic diversity across all the breeds. Estimates of mean allele number and observed heterozygosity across all loci and population were 8.784 ± 0.25 and 0.653 ± 0.014, respectively. Differences among breeds accounted for 13.3 % of total genetic variability. Despite high genetic diversity, significant inbreeding was also observed within eight populations. Genetic distances and cluster analysis showed a close relationship between breeds according to proximity in geographic distribution. The genetic distance, STRUCTURE and Principal Coordinate Analysis concluded that the Southern Indian Ongole cattle are the most distinct among the investigated cattle populations. Sequencing of hypervariable mitochondrial DNA region on a subset of 170 cattle revealed sixty haplotypes with haplotypic diversity of 0.90240, nucleotide diversity of 0.02688 and average number of nucleotide differences as 6.07407. Two major star clusters for haplotypes indicated population expansion for Indian cattle. Nuclear and mitochondrial genomes show a similar pattern of genetic variability and genetic differentiation. Various analyses concluded that the Southern breed ‘Ongole’ was distinct from breeds of Northern/ Central India. Overall these results provide basic information about genetic diversity and structure of Indian cattle which should have implications for management and conservation of indicine cattle diversity.

Development of maizeSNP3072, a high-throughput compatible SNP array, for DNA fingerprinting identification of Chinese maize varieties.

Abstract: Single nucleotide polymorphisms (SNPs) are abundant and evenly distributed throughout the maize (Zea mays L.) genome. SNPs have several advantages over simple sequence repeats, such as ease of data comparison and integration, high-throughput processing of loci, and identification of associated phenotypes. SNPs are thus ideal for DNA fingerprinting, genetic diversity analysis, and marker-assisted breeding. Here, we developed a high-throughput and compatible SNP array, maizeSNP3072, containing 3072 SNPs developed from the maizeSNP50 array. To improve genotyping efficiency, a high-quality cluster file, maizeSNP3072_GT.egt, was constructed. All 3072 SNP loci were localized within different genes, where they were distributed in exons (43 %), promoters (21 %), 3′ untranslated regions (UTRs; 22 %), 5′ UTRs (9 %), and introns (5 %). The average genotyping failure rate using these SNPs was only 6 %, or 3 % using the cluster file to call genotypes. The genotype consistency of repeat sample analysis on Illumina GoldenGate versus Infinium platforms exceeded 96.4 %. The minor allele frequency (MAF) of the SNPs averaged 0.37 based on data from 309 inbred lines. The 3072 SNPs were highly effective for distinguishing among 276 examined hybrids. Comparative analysis using Chinese varieties revealed that the 3072SNP array showed a better marker success rate and higher average MAF values, evaluation scores, and variety-distinguishing efficiency than the maizeSNP50K array. The maizeSNP3072 array thus can be successfully used in DNA fingerprinting identification of Chinese maize varieties and shows potential as a useful tool for germplasm resource evaluation and molecular marker-assisted breeding. Electronic supplementary material The online version of this article (doi:10.1007/s11032-015-0335-0) contains supplementary material, which is available to authorized users.

Population structure and genetic diversity characterization of a sunflower association mapping population using SSR and SNP markers

Abstract: Argentina has a long tradition of sunflower breeding, and its germplasm is a valuable genetic resource worldwide. However, knowledge of the genetic constitution and variability levels of the Argentinean germplasm is still scarce, rendering the global map of cultivated sunflower diversity incomplete. In this study, 42 microsatellite loci and 384 single nucleotide polymorphisms (SNPs) were used to characterize the first association mapping population used for quantitative trait loci mapping in sunflower, along with a selection of allied open-pollinated and composite populations from the germplasm bank of the National Institute of Agricultural Technology of Argentina. The ability of different kinds of markers to assess genetic diversity and population structure was also evaluated. The analysis of polymorphism in the set of sunflower accessions studied here showed that both the microsatellites and SNP markers were informative for germplasm characterization, although to different extents. In general, the estimates of genetic variability were moderate. The average genetic diversity, as quantified by the expected heterozygosity, was 0.52 for SSR loci and 0.29 for SNPs. Within SSR markers, those derived from non-coding regions were able to capture higher levels of diversity than EST-SSR. A significant correlation was found between SSR and SNP- based genetic distances among accessions. Bayesian and multivariate methods were used to infer population structure. Evidence for the existence of three different genetic groups was found consistently across data sets (i.e., SSR, SNP and SSR + SNP), with the maintainer/restorer status being the most prevalent characteristic associated with group delimitation. The present study constitutes the first report comparing the performance of SSR and SNP markers for population genetics analysis in cultivated sunflower. We show that the SSR and SNP panels examined here, either used separately or in conjunction, allowed consistent estimations of genetic diversity and population structure in sunflower breeding materials. The generated knowledge about the levels of diversity and population structure of sunflower germplasm is an important contribution to this crop breeding and conservation.

Evaluation of marker-assisted selection for the stripe rust resistance gene Yr15, introgressed from wild emmer wheat.

Abstract: Stripe rust disease is caused by the fungus Puccinia striiformis f. sp. tritici and severely threatens wheat worldwide, repeatedly breaking resistance conferred by resistance genes and evolving more aggressive strains. Wild emmer wheat, Triticum dicoccoides, is an important source for novel stripe rust resistance (Yr) genes. Yr15, a major gene located on chromosome 1BS of T. dicoccoides, was previously reported to confer resistance to a broad spectrum of stripe rust isolates, at both seedling and adult plant stages. Introgressions of Yr15 into cultivated T. aestivum bread wheat and T. durum pasta wheat that began in the 1980s are widely used. In the present study, we aimed to validate SSR markers from the Yr15 region as efficient tools for marker-assisted selection (MAS) for introgression of Yr15 into wheat and to compare the outcome of gene introgression by MAS and by conventional phenotypic selection. Our findings establish the validity of MAS for introgression of Yr15 into wheat. We show that the size of the introgressed segment, defined by flanking markers, varies for both phenotypic selection and MAS. The genetic distance of the MAS marker from Yr15 and the number of backcross steps were the main factors affecting the length of the introgressed donor segments. Markers Xbarc8 and Xgwm493, which are the nearest flanking markers studied, were consistent and polymorphic in all 34 introgressions reported here and are therefore the most recommended markers for the introgression of Yr15 into wheat cultivars. Introgression directed by markers, rather than by phenotype, will facilitate simultaneous selection for multiple stripe rust resistant genes and will help to avoid escapees during the selection process.

Second-Generation Linkage Maps for the Pacific Oyster Crassostrea gigas Reveal Errors in Assembly of Genome Scaffolds.

Abstract: The Pacific oyster Crassostrea gigas, a widely cultivated marine bivalve mollusc, is becoming a genetically and genomically enabled model for highly fecund marine metazoans with complex life-histories. A genome sequence is available for the Pacific oyster, as are first-generation, low-density, linkage and gene-centromere maps mostly constructed from microsatellite DNA markers. Here, higher density, second-generation, linkage maps are constructed from more than 1100 coding (exonic) single-nucleotide polymorphisms (SNPs), as well as 66 previously mapped microsatellite DNA markers, all typed in five families of Pacific oysters (nearly 172,000 genotypes). The map comprises 10 linkage groups, as expected, has an average total length of 588 cM, an average marker-spacing of 1.0 cM, and covers 86% of a genome estimated to be 616 cM. All but seven of the mapped SNPs map to 618 genome scaffolds; 260 scaffolds contain two or more mapped SNPs, but for 100 of these scaffolds (38.5%), the contained SNPs map to different linkage groups, suggesting widespread errors in scaffold assemblies. The 100 misassembled scaffolds are significantly longer than those that map to a single linkage group. On the genetic maps, marker orders and intermarker distances vary across families and mapping methods, owing to an abundance of markers segregating from only one parent, to widespread distortions of segregation ratios caused by early mortality, as previously observed for oysters, and to genotyping errors. Maps made from framework markers provide stronger support for marker orders and reasonable map lengths and are used to produce a consensus high-density linkage map containing 656 markers.

Evaluation of microsatellite sequence-tagged site markers for characterizing Vitis vinifera cultivars.

Abstract: Twenty four cultivars and clones from Italian germplasm collections were DNA typed for the four sequence-tagged microsatellite sites VVS1-FP, VVS2-JOE, VVS5-FP and VV29-TAM. The analysis was performed using primers labelled with different fluorochromes and using a GENESCAN apparatus. The results were compared with data obtained for samples from Australian collections. By comparing the combined genotype of the studied loci all the cultivars could be singularly distinguished except Favorita, Pigato and Vermentino, thus supporting the hypothesis that all three are the same cultivar. Cultivars common to Italian and Australian germplasm collections were found to have the same genotype indicating that the study of microsatellites as sequence-tagged site (STS) markers is a suitable universal system for worldwide grapevine cultivar identification. Application of the semi-automated GENESCAN system made the analysis of microsatellite STS markers fast and reproducible between laboratories.

Ultraconserved Elements Sequencing as a Low-Cost Source of Complete Mitochondrial Genomes and Microsatellite Markers in Non-Model Amniotes

Abstract: Sequence capture of ultraconserved elements (UCEs) associated with massively parallel sequencing has become a common source of nuclear data for studies of animal systematics and phylogeography. However, mitochondrial and microsatellite variation are still commonly used in various kinds of molecular studies, and probably will complement genomic data in years to come. Here we show that besides providing abundant genomic data, UCE sequencing is an excellent source of both sequences for microsatellite loci design and complete mitochondrial genomes with high sequencing depth. Identification of dozens of microsatellite loci and assembly of complete mitogenomes is exemplified here using three species of Poospiza warbling finches from southern and southeastern Brazil. This strategy opens exciting opportunities to simultaneously analyze genome-wide nuclear datasets and traditionally used mtDNA and microsatellite markers in non-model amniotes at no additional cost.

Construction of a SNP-based high-density genetic map for pummelo using RAD sequencing.

Abstract: Pummelo (Citrus grandis) is one of the most important gene pools for citrus breeding programmes. A high-density linkage map is a valuable tool for functional genomics and genetic breeding studies. A newly developed genome sequence-based marker technology, restriction site-associated DNA (RAD) sequencing, has proven to be powerful for the rapid discovery and genotyping of genome-wide SNP markers and for the high-density genetic map construction. We present the construction of a high-density genetic map of pummelo using RAD sequencing. An F1 population of 124 individuals and its parents (‘Pingshan’ pummelo and ‘Guanxi’ pummelo) were applied to the map construction. One thousand five hundred forty three high-quality single nucleotide polymorphism (SNP) markers were developed and mapped to nine linkage groups. In addition, 20 simple sequence repeat (SSR) markers were included and showed general consistency with the SNP markers. These 1563 markers constituted a total genetic length of 976.58 cM and an average of 0.62 cM between adjacent loci. The number of markers within each linkage group (LG) ranged from 81 (for LG4) to 285 (for LG2). A comparison of the genetic maps to the published sweet orange (Citrus sinensis) genome revealed both conservation and variations. The alignment of the LGs from this map was also shown in comparison with a previously genetic linkage map from pummelo. This study showed that the RAD sequencing allowed the rapid discovery of a large number of SNPs in the pummelo. The SNP-based high-density genetic map for pummelo was successfully generated by using these designed SNP markers. The completed genetic map is a valuable resource for further pummelo genetic studies and provides useful information for gene positional cloning, MAS breeding and C. grandis genome assembly.

Diversity in the breadfruit complex ( Artocarpus , Moraceae): genetic characterization of critical germplasm

Abstract: Breadfruit (Artocarpus altilis, Moraceae) is a traditional staple crop in Oceania and has been introduced throughout the tropics. This study examines important germplasm collections of breadfruit and its closest wild relatives and aims to (1) characterize genetic diversity, including identification of unknown and duplicate accessions, (2) evaluate genetic structure and hybridization within the breadfruit complex, and (3) compare utility of microsatellite markers to previously reported amplified fragment length polymorphism (AFLP) and isozyme markers in differentiating among cultivars. Data for 19 microsatellite loci were collected for 349 individuals (representing 255 accessions) including breadfruit (A. altilis), two wild relatives (Artocarpus camansi and Artocarpus mariannensis), and putative hybrids (A. altilis × A. mariannensis). Accessions were of mixed ploidy and regional origin, but predominantly from Oceania. Microsatellite loci collectively had a polymorphic information content (PIC) of 0.627 and distinguished 197 unique genotypes sorted into 129 different lineages, but a single genotype accounts for 49 % of all triploid breadfruit examined. Triploid hybrids and diploid A. altilis exhibited the highest levels of diversity as measured by allele number and gene diversity. Most accessions (75 %) of unknown origin matched either a known genotype or lineage group in the collection. Putative hybrids all had genetic contributions from A. mariannensis but ranged in the level of genetic contribution from A. altilis. Microsatellite markers were found to be more informative than isozyme markers and slightly less informative, with regard to accession discrimination, than AFLP markers. This set of microsatellite markers and the dataset presented here will be valuable for breadfruit germplasm management and conservation.

Characterization of Iranian grapevine cultivars using microsatellite markers

Abstract: Sixty-two grapevine ( Vitis spp.) accessions from Iran and the USA were characterized at 9 highly polymorphic microsatellite loci using fluorescent primers and a capillary electrophoresis fragment sizing system. The number of alleles observed per locus ranged from 4 to 16 and heterozygosity values ranged from 0.47 to 0.86. Genetic similarity was estimated for each pair of accessions as the proportion of shared alleles. A phenogram constructed from genetic dissimilarity values revealed three clusters, one each for table grapes, wine grapes and rootstocks. The phenogram also revealed three clonal sets (Askari, Bidane and Yaghoti) as well as some synonyms and homonyms among Iranian table grape cultivars.

Simple Sequence Repeat Markers in Genetic Divergence and Marker-Assisted Selection of Rice Cultivars: A Review

Abstract: Sequencing of rice genome has facilitated the understanding of rice evolution and has been utilized extensively for mining of DNA markers to facilitate marker-assisted breeding. Simple sequence repeat (SSR) markers that are tandemly repeated nucleotide sequence motifs flanked by unique sequences are presently the maker of choice in rice improvement due to their abundance, co-dominant inheritance, high levels of allelic diversity, and simple reproducible assay. The current level of genome coverage by SSR markers in rice is sufficient to employ them for genotype identification and marker-assisted selection in breeding for mapping of genes and quantitative trait loci analysis. This review provides comprehensive information on the mapping and applications of SSR markers in investigation of rice cultivars to study their genetic divergence and marker-assisted selection of important agronomic traits.

Genome-wide survey and analysis of microsatellites in giant panda ( Ailuropoda melanoleuca ), with a focus on the applications of a novel microsatellite marker system

Abstract: The giant panda (Ailuropoda melanoleuca) is a critically endangered species endemic to China Microsatellites have been preferred as the most popular molecular markers and proven effective in estimating population size, paternity test, genetic diversity for the critically endangered species The availability of the giant panda complete genome sequences provided the opportunity to carry out genome-wide scans for all types of microsatellites markers, which now opens the way for the analysis and development of microsatellites in giant panda By screening the whole genome sequence of giant panda in silico mining, we identified microsatellites in the genome of giant panda and analyzed their frequency and distribution in different genomic regions Based on our search criteria, a repertoire of 855,058 SSRs was detected, with mono-nucleotides being the most abundant SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions A total of 160 primer pairs were designed to screen for polymorphic microsatellites using the selected tetranucleotide microsatellite sequences The 51 novel polymorphic tetranucleotide microsatellite loci were discovered based on genotyping blood DNA from 22 captive giant pandas in this study Finally, a total of 15 markers, which showed good polymorphism, stability, and repetition in faecal samples, were used to establish the novel microsatellite marker system for giant panda Meanwhile, a genotyping database for Chengdu captive giant pandas (n = 57) were set up using this standardized system What’s more, a universal individual identification method was established and the genetic diversity were analysed in this study as the applications of this marker system The microsatellite abundance and diversity were characterized in giant panda genomes A total of 154,677 tetranucleotide microsatellites were identified and 15 of them were discovered as the polymorphic and stable loci The individual identification method and the genetic diversity analysis method in this study provided adequate material for the future study of giant panda

Genome-Wide Mining, Characterization and Development of Microsatellite Markers in Gossypium Species

Abstract: Although much research has been conducted to characterize microsatellites and develop markers, the distribution of microsatellites remains ambiguous and the use of microsatellite markers in genomic studies and marker-assisted selection is limited. To identify microsatellites for cotton research, we mined 100,290, 83,160, and 56,937 microsatellites with frequencies of 41.2, 49.1, and 74.8 microsatellites per Mb in the recently sequenced Gossypium species: G. hirsutum, G. arboreum, and G. raimondii, respectively. The distributions of microsatellites in their genomes were non-random and were positively and negatively correlated with genes and transposable elements, respectively. Of the 77,996 developed microsatellite markers, 65,498 were physically anchored to the 26 chromosomes of G. hirsutum with an average marker density of 34 markers per Mb. We confirmed 67,880 (87%) universal and 7,705 (9.9%) new genic microsatellite markers. The polymorphism was estimated in above three species by in silico PCR and validated with 505 markers in G. hirsutum. We further predicted 8,825 polymorphic microsatellite markers within G. hirsutum acc. TM-1 and G. barbadense cv. Hai7124. In our study, genome-wide mining and characterization of microsatellites, and marker development were very useful for the saturation of the allotetraploid genetic linkage map, genome evolution studies and comparative genome mapping.

Genome-wide development of novel miRNA-based microsatellite markers of rice ( Oryza sativa ) for genotyping applications

Abstract: Rice (Oryza sativa L.) plays a crucial role in assuring food security to the world’s population and is recognized as a superior model plant of monocot crops. Molecular markers, especially co-dominant markers such as simple sequence repeats (SSRs), play an important role in marker-assisted breeding of rice. Although a large number of SSRs are available, most of them are either from protein-coding regions or untranslated regions of the rice genome. SSRs from non-coding regions of the rice genome are almost undiscovered, but discovery of new SSR markers remains a challenge for molecular breeders. The development of novel markers from the conserved regions of different genomes will thus be useful for studying the genetic diversity of closely related species or self-pollinated species. In the present study, a genome-wide investigation yielded a total of 129 SSR markers from the conserved microRNA (miRNA) genes of rice, and 20 of them with repeat numbers ≥7, from all the 12 chromosomes, were validated among 24 diverse rice genotypes. The highest number of repeat motifs corresponded to di-nucleotides, while the tetra-nucleotides were repeated the lowest number of times. Our results therefore show that the number of repeats is inversely related to the length of repeat. The genetic diversity was found to be higher among the exotic material than the Indian landraces, as indicated by their polymorphic information content values of 0.31 and 0.29, respectively. The results hence showed that the miRNA-based microsatellite marker system is a very proficient, novel and breeder-friendly source for genetic diversity analysis or genotyping of rice. The present study is the first report on genome-wide identification and characterization of rice miRNA-SSRs.

Effect of microsatellite selection on individual and population genetic inferences: an empirical study using cross-specific and species-specific amplifications.

Abstract: Although whole-genome sequencing is becoming more accessible and feasible for nonmodel organisms, microsatellites have remained the markers of choice for various population and conservation genetic studies. However, the criteria for choosing microsatellites are still controversial due to ascertainment bias that may be introduced into the genetic inference. An empirical study of red deer (Cervus elaphus) populations, in which cross-specific and species-specific microsatellites developed through pyrosequencing of enriched libraries, was performed for this study. Two different strategies were used to select the species-specific panels: randomly vs. highly polymorphic markers. The results suggest that reliable and accurate estimations of genetic diversity can be obtained using random microsatellites distributed throughout the genome. In addition, the results reinforce previous evidence that selecting the most polymorphic markers leads to an ascertainment bias in estimates of genetic diversity, when compared with randomly selected microsatellites. Analyses of population differentiation and clustering seem less influenced by the approach of microsatellite selection, whereas assigning individuals to populations might be affected by a random selection of a small number of microsatellites. Individual multilocus heterozygosity measures produced various discordant results, which in turn had impacts on the heterozygosity-fitness correlation test. Finally, we argue that picking the appropriate microsatellite set should primarily take into account the ecological and evolutionary questions studied. Selecting the most polymorphic markers will generally overestimate genetic diversity parameters, leading to misinterpretations of the real genetic diversity, which is particularly important in managed and threatened populations.

Grapevine fingerprinting using microsatellite repeats

Abstract: Five different microsatellite loci have been amplified through PCR (Polymerase Chain Reaction) in order to find polymorphisms useful for identification of 16 cvs and 42 clones or populations of Vitis vinifera L. No genetic variability was detected among clones and populations. Ail the cultivars have been identified, except those derived from bud mutations involving the colour of the berries, as the Pinot group (P. noir, P. blanc, P. gris), or those thought to be closely correlated or synonymous, such as 'Refosco di Faedis' and 'Refoscone'. One locus (VVS2) showed a high polymorphism with 12 alleles detected on high resolution agarose gel.

Developing genome-wide microsatellite markers of bamboo and their applications on molecular marker assisted taxonomy for accessions in the genus Phyllostachys

Abstract: Morphology-based taxonomy via exiguously reproductive organ has severely limitation on bamboo taxonomy, mainly owing to infrequent and unpredictable flowering events of bamboo. Here, we present the first genome-wide analysis and application of microsatellites based on the genome of moso bamboo (Phyllostachys edulis) to assist bamboo taxonomy. Of identified 127,593 microsatellite repeat-motifs, the primers of 1,451 microsatellites were designed and 1,098 markers were physically mapped on the genome of moso bamboo. A total of 917 markers were successfully validated in 9 accessions with ~39.8% polymorphic potential. Retrieved from validated microsatellite markers, 23 markers were selected for polymorphic analysis among 78 accessions and 64 alleles were detected with an average of 2.78 alleles per primers. The cluster result indicated the majority of the accessions were consistent with their current taxonomic classification, confirming the suitability and effectiveness of the developed microsatellite markers. The variations of microsatellite marker in different species were confirmed by sequencing and in silico comparative genome mapping were investigated. Lastly, a bamboo microsatellites database (http://www.bamboogdb.org/ssr) was implemented to browse and search large information of bamboo microsatellites. Consequently, our results of microsatellite marker development are valuable for assisting bamboo taxonomy and investigating genomic studies in bamboo and related grass species.

A Set of Cytogenetic Markers Allows the Precise Identification of All A-Genome Chromosomes in Diploid and Polyploid Wheat.

Abstract: Karyotypes of 3 diploid wheat species containing different variants of the A-genome, Triticum boeoticum (A b ), T. monococcum (A b ), and T. urartu (A u ), were examined using C-banding and FISH with DNA probes representing 5S and 45S rDNA families, the microsatellite sequences GAA n and GTT n , the already known satellite sequences pSc119.2, Spelt52, Fat , pAs1, and pTa535, and a newly identified repeat called Aesp_SAT86. The C-banding patterns of the 3 species in general were similar; differences were observed in chromosomes 4A and 6A. Besides 2 major 45S rDNA loci on chromosomes 1A and 5A, 2 minor polymorphic NORs were observed in the terminal part of 5AL and in the distal part of 6AS in all species. An additional minor locus was found in the distal part of 7A b L of T. boeoticum and T. monococcum, but not in T. urartu. Two 5S rDNA loci were observed in 1AS and 5AS.

Toll-like receptor diversity in 10 threatened bird species: relationship with microsatellite heterozygosity

Abstract: Measuring individual-level heterozygosity in threatened species is one approach to understanding and mitigating losses of genetic diversity and the role of inbreeding depression in those populations In many conservation contexts, this goal is approached by assaying levels of microsatellite diversity, and inference is often extended to functional genomic regions Our study quantifies diversity of innate immunity toll-like receptor (TLR) genes in 10 threatened New Zealand birds across four avian orders, with an average of 201 individuals and 62 TLR loci (sequences averaging 850 bp in length) per species We provide detailed TLR diversity statistics for these 10 species, which showed more evidence for genetic drift than balancing selection at TLR loci, with two possible exceptions (TLR1LA for hihi and TLR5 for kokako) Our observations also support a possible gene-duplication of TLR7 in rock wren, indicating that a TLR7 duplication previously observed in other passerines may have occurred early in the divergence of this order In addition to these analyses of population-level TLR sequence diversity, we used an average of 146 polymorphic microsatellite loci per species to study, for the first time, the relationship between microsatellite internal relatedness (a measure of individual homozygosity) and TLR heterozygosity There was no relationship between microsatellite and TLR heterozygosity of individuals within species, suggesting that the predictive power of microsatellites to evaluate functional diversity is poor, and highlighting the value of adding data from putatively functional genomic regions, such as TLRs, in the study of genetic diversity of threatened species Overall this study provides valuable data for comparison with more widespread species, and facilitates research into the importance of TLR diversity in natural populations of conservation concern

Impact of repetitive DNA on sex chromosome evolution in plants.

Abstract: Structurally and functionally diverged sex chromosomes have evolved in many animals as well as in some plants. Sex chromosomes represent a specific genomic region(s) with locally suppressed recombination. As a consequence, repetitive sequences involving transposable elements, tandem repeats (satellites and microsatellites), and organellar DNA accumulate on the Y (W) chromosomes. In this paper, we review the main types of repetitive elements, their gathering on the Y chromosome, and discuss new findings showing that not only accumulation of various repeats in non-recombining regions but also opposite processes form Y chromosome. The aim of this review is also to discuss the mechanisms of repetitive DNA spread involving (retro) transposition, DNA polymerase slippage or unequal crossing-over, as well as modes of repeat removal by ectopic recombination. The intensity of these processes differs in non-recombining region(s) of sex chromosomes when compared to the recombining parts of genome. We also speculate about the relationship between heterochromatinization and the formation of heteromorphic sex chromosomes.

Genome-wide identification of SSR and SNP markers from the non-heading Chinese cabbage for comparative genomic analyses

Abstract: Non-heading Chinese cabbage (NHCC), belonging to Brassica, is an important leaf vegetable in Asia. Although genetic analyses have been performed through conventional selection and breeding efforts, the domestication history of NHCC and the genetics underlying its morphological diversity remain unclear. Thus, the reliable molecular markers representative of the whole genome are required for molecular-assisted selection in NHCC. A total of 20,836 simple sequence repeats (SSRs) were detected in NHCC, containing repeat types from mononucleotide to nonanucleotide. The average density was 62.93 SSRs/Mb. In gene regions, 5,435 SSRs were identified in 4,569 genes. A total of 5,008 primer pairs were designed, and 74 were randomly selected for validation. Among these, 60 (81.08%) were polymorphic in 18 Cruciferae. The number of polymorphic bands ranged from two to five, with an average of 2.70 for each primer. The average values of the polymorphism information content, observed heterozygosity, Hardy-Weinberg equilibrium, and Shannon’s information index were 0.2970, 0.4136, 0.5706, and 0.5885, respectively. Four clusters were classified according to the unweighted pair-group method with arithmetic average cluster analysis of 18 genotypes. In addition, a total of 1,228,979 single nucleotide polymorphisms (SNPs) were identified in the NHCC through a comparison with the genome of Chinese cabbage, and the average SNP density in the whole genome was 4.33/Kb. The number of SNPs ranged from 341,939 to 591,586 in the 10 accessions, and the average heterozygous SNPs ratio was ~42.53%. All analyses showed these markers were high quality and reliable. Therefore, they could be used in the construction of a linkage map and for genetic diversity studies for NHCC in future. This is the first systematic and comprehensive analysis and identification of SSRs in NHCC and 17 species. The development of a large number of SNP and SSR markers was successfully achieved for NHCC. These novel markers are valuable for constructing genetic linkage maps, comparative genome analysis, quantitative trait locus (QTL) mapping, genome-wide association studies, and marker-assisted selection in NHCC breeding system research.

Efficient development of highly polymorphic microsatellite markers based on polymorphic repeats in transcriptome sequences of multiple individuals.

Abstract: The first hurdle in developing microsatellite markers, cloning, has been overcome by next generation sequencing. The second hurdle is testing to differentiate polymorphic from non-polymorphic loci. The third hurdle, somewhat hidden, is that only polymorphic markers with a large effective number of alleles are sufficiently informative to be deployed in multiple studies. Both steps are laborious and still done manually. We have developed a strategy in which we first screen reads from multiple genotypes for repeats that show the most length variants, and only these are subsequently developed into markers. We validated our strategy in tetraploid garden rose using Illumina paired-end transcriptome sequences of 11 roses. Out of 48 tested two markers failed to amplify but all others were polymorphic. Ten loci amplified more than one locus, indicating duplicated genes or gene families. Completely avoiding duplicated loci will be difficult because the range of numbers of predicted alleles of highly polymorphic single- and multi-locus markers largely overlapped. Of the remainder, half were replicate markers (i.e., multiple primer pairs for one locus), indicating the difficulty of correctly filtering short reads containing repeat sequences. We subsequently refined the approach to eliminate multiple primer sets to the same loci. The remaining 18 markers were all highly polymorphic, amplifying on average 11.7 alleles per marker (range = 6 to 20) in 11 tetraploid roses, exceeding the 8.2 alleles per marker of the 24 most polymorphic markers genotyped previously. This strategy, therefore, represents a major step forward in the development of highly polymorphic microsatellite markers.

Construction of a High-Density Simple Sequence Repeat Consensus Genetic Map for Pear (Pyrus spp.)

Abstract: As an important fruit crop that is widely grown commercially in temperate regions of the world, pear (Pyrus) is a target for pursuing efficient breeding strategies. Construction of a reliable and dense genetic linkage map is critical for undertaking marker-assisted breeding. In this study, a population of 56 F1 seedlings of ‘Bayuehong’ × ‘Dangshansuli’ was used to construct a high-density simple sequence repeat (SSR)-based genetic linkage map. A total of 1,756 SSR markers, including 1,341 newly designed SSRs based on whole-genome sequencing of an Asiatic pear along with 415 previously reported SSRs, were first evaluated for polymorphism. Based on 894 SSRs demonstrating polymorphism, a consensus genetic map consisting of 734 loci distributed along all 17 linkage groups (LG) was constructed, with a total length of 1,661.4 cM and with an average marker interval of 2.26 cM. Comparisons among different maps of pear and apple were then made based on positions of previously mapped SSR markers on the consensus map. As a result, homologous linkage groups LG3 and LG11, LG5 and LG10, LG9 and LG17, LG13 and LG16, LG8 and LG15 have been identified. This high-density SSR map along with a set of SSR markers covering the whole genome of pear will greatly facilitate integration of independent maps, aid in pursuing comparative genome studies, and in evaluation of different germplasm in future genetic and breeding studies.

Identification and development of new polymorphic microsatellite markers using genome assembly for Ganoderma boninense, causal agent of oil palm basal stem rot disease

Abstract: Ganoderma boninense is a telluric lignicolous basidiomycete and the causal agent of basal stem rot, one of the most devastating diseases of the oil palm (Elaeis guineensis). While the fight against G. boninense is of major concern in Southeast Asia, little information is available regarding the genetic diversity and evolutionary history of the fungus. In this context, the development of an informative molecular marker set to characterize the diversity of G. boninense is a key step towards understanding the biology of this pathogen. A G. boninense draft genome sequence of 63 Mbp, assembled using 454 and Illumina sequencing technology, was used to identify and develop a set of microsatellite markers (simple sequence repeats, SSRs). A total of 2487 SSRs were identified, for which 145 SSR primer pairs were designed. These SSRs are characterized by di- to hexanucleotide motifs with 5 to 34 repetitions. Ninety-seven SSR loci were successfully amplified on an initial small set of G. boninense isolates from Indonesia. A collection of 107 isolates from several regions in Southeast Asia were screened to characterize each locus for allele number, polymorphism information criterion and the presence or absence of null alleles at each locus. These results allowed us to propose an effective set of 17 SSRs for studying genetic diversity within G. boninense.